Rationally Designed Peptoid Inhibitors of Amyloid-ß Oligomerization.

While plaques comprised of fibrillar Aß aggregates are hallmarks of Alzheimer's disease, soluble Aß oligomers present higher neurotoxicity. Thus, one therapeutic approach is to prevent the formation of Aß oligomers and reduce their associated harmful effects. We have proposed a peptoid mimic of the Aß hydrophobic KLVFF core as an ideal candidate aggregation inhibitor due to its ability to evade proteolytic degradation via repositioning of the side chain from the α-carbon to the amide nitrogen. This peptoid, JPT1, utilizes chiral sidechains to achieve a helical structure, while C-terminal addition of two phenylalanine residues places aromatic groups on two sides of the helix with spacing designed to facilitate interaction with amyloid ß-sheet structure. We have previously shown that JPT1 modulates Aß fibril formation. Here, we demonstrate that JPT1 also modulates Aß oligomerization, and we explore the role of the charge on the linker between the KLVFF mimic and the extended aromatic residues. Additionally, we demonstrate that peptoid-induced changes in Aß oligomerization correlate with attenuation of oligomer-induced nuclear factor-κB activation in SH-SY5Y human neuroblastoma cells. These findings support the therapeutic potential of peptoids to target early stages of Aß aggregation and impact the associated Aß-induced cellular response.

Conformational Studies of ß-Azapeptoid Foldamers: A New Class of Peptidomimetics with Confined Dihedrals.

Controlling amide bond geometries and the secondary structures of ß-peptoids is a challenging task as they contain several rotatable single bonds in their backbone. Herein, we describe the synthesis and conformational properties of novel "ß-azapeptoids" with confined dihedrals. We discuss how the acylhydrazide sidechains in these molecules enforce trans amide geometries (ω ~180°) via steric and stereoelectronic effects. We also show that the Θ(Cα -Cß ) and Ψ(OC-Cα ) backbone torsions of ß-azapeptoids occupy a narrow range (170-180°) that can be rationalized by the staggered conformational preference of the backbone methylene carbons and a novel backbone nO →σ*Cß-N interaction discovered in this study. However, the ϕ (Cß -N) torsion remains freely rotatable and, depending on ϕ, the sidechains can be parallel, perpendicular, and anti-parallel relative to each other. In fact, we observed parallel and perpendicular relative orientations of sidechains in the crystal geometries of ß-azapeptoid dimers. We show that ϕ of ß-azapeptoids can be controlled by incorporating a bulky substituent at the backbone ß-carbon, which could provide complete control over all the backbone dihedrals. Finally, we show that the ϕ and Ψ dihedrals of ß-azapeptoids resemble that of a PPII helix and they retain PPII structure when incorporated in Host-guest proline peptides.

Anti-Neuroinflammatory Effects of a Macrocyclic Peptide-Peptoid Hybrid in Lipopolysaccharide-Stimulated BV2 Microglial Cells.

Inflammation processes of the central nervous system (CNS) play a vital role in the pathogenesis of several neurological and psychiatric disorders like depression. These processes are characterized by the activation of glia cells, such as microglia. Clinical studies showed a decrease in symptoms associated with the mentioned diseases after the treatment with anti-inflammatory drugs. Therefore, the investigation of novel anti-inflammatory drugs could hold substantial potential in the treatment of disorders with a neuroinflammatory background. In this in vitro study, we report the anti-inflammatory effects of a novel hexacyclic peptide-peptoid hybrid in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The macrocyclic compound X15856 significantly suppressed Interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), c-c motif chemokine ligand 2 (CCL2), CCL3, C-X-C motif chemokine ligand 2 (CXCL2), and CXCL10 expression and release in LPS-treated BV2 microglial cells. The anti-inflammatory effects of the compound are partially explained by the modulation of the phosphorylation of p38 mitogen-activated protein kinases (MAPK), p42/44 MAPK (ERK 1/2), protein kinase C (PKC), and the nuclear factor (NF)-κB, respectively. Due to its remarkable anti-inflammatory properties, this compound emerges as an encouraging option for additional research and potential utilization in disorders influenced by inflammation, such as depression.

A peptoid-based inhibitor of protein arginine methyltransferase 1 (PRMT1) induces apoptosis and autophagy in cancer cells.

Protein arginine methyltransferases (PRMTs) are S-adenosylmethionine-dependent enzymes that transfer a methyl group to arginine residues within proteins, most notably histones. The nine characterized PRMT family members are divided into three types depending on the resulting methylated product: asymmetric dimethylarginine (Type I PRMT), symmetric dimethylarginine (Type II PRMT), or monomethylated arginine (Type III PRMT). In some cancers, the resulting product can lead to either increased or decreased transcription of cancer-related genes, suggesting PRMT family members may be valid therapeutic targets. Traditionally, peptide-based compounds have been employed to target this family of enzymes, which has resulted in multiple tool and lead compounds being developed. However, peptide-based therapeutics suffer from poor stability and short half-lives, as proteases can render them useless by hydrolytic degradation. Conversely, peptoids, which are peptide-mimetics composed of N-substituted glycine monomers, are less susceptible to hydrolysis, resulting in improved stability and longer half-lives. Herein, we report the development of a bioavailable, peptoid-based PRMT1 inhibitor that induces cell death in MDA468 and HCT116 cancer cell lines while not exhibiting any significant impact on nontumorigenic HepaRG or normal human mammary epithelial cells. Furthermore, the inhibitor described herein appears to induce both apoptosis and autophagy, suggesting it may be a less toxic cytostatic agent. In conclusion, we propose this peptoid-based inhibitor has significant anticancer and therapeutic potential by reducing cell viability, growth, and size in breast and colon cancer. Further experimentation will help determine the mechanism of action and downstream effects of this compound.

Co-Assembly of Carbon Nanotube Porins into Biomimetic Peptoid Membranes.

Robust and cost-effective membrane-based separations are essential to solving many global crises, such as the lack of clean water. Even though the current polymer-based membranes are widely used for separations, their performance and precision can be enhanced by using a biomimetic membrane architecture that consists of highly permeable and selective channels embedded in a universal membrane matrix. Researchers have shown that artificial water and ion channels, such as carbon nanotube porins (CNTPs), embedded in lipid membranes can deliver strong separation performance. However, their applications are limited by the relative fragility and low stability of the lipid matrix. In this work, we demonstrate that CNTPs can co-assemble into two dimension (2D) peptoid membrane nanosheets, opening up a way to produce highly programmable synthetic membranes with superior crystallinity and robustness. A combination of molecular dynamics (MD) simulations, Raman spectroscopy, X-ray diffraction (XRD), and atomic force microscopy (AFM) measurements to verify the co-assembly of CNTP and peptoids are used and show that it does not disrupt peptoid monomer packing within the membrane. These results provide a new option for designing affordable artificial membranes and highly robust nanoporous solids.

Modulating the RAGE-Induced Inflammatory Response: Peptoids as RAGE Antagonists.

While the primary pathology of Alzheimer's disease (AD) is defined by brain deposition of amyloid-ß (Aß) plaques and tau neurofibrillary tangles, chronic inflammation has emerged as an important factor in AD etiology. Upregulated cell surface expression of the receptor for advanced glycation end-products (RAGE), a key receptor of innate immune response, is reported in AD. In parallel, RAGE ligands, including Aß aggregates, HMGB1, and S100B, are elevated in AD brain. Activation of RAGE by these ligands triggers release of inflammatory cytokines and upregulates cell surface RAGE. Despite such observation, there are currently no therapeutics that target RAGE for treatment of AD-associated neuroinflammation. Peptoids, a novel class of potential AD therapeutics, display low toxicity, facile blood-brain barrier permeability, and resistance to proteolytic degradation. In the current study, peptoids were designed to mimic Aß, a ligand that binds the V-domain of RAGE, and curtail RAGE inflammatory activation. We reveal the nanomolar binding capability of peptoids JPT1 and JPT1a to RAGE and demonstrate their ability to attenuate lipopolysaccharide-induced pro-inflammatory cytokine production as well as upregulation of RAGE cell surface expression. These results support RAGE antagonist peptoid-based mimics as a prospective therapeutic strategy to counter neuroinflammation in AD and other neurodegenerative diseases.

Site Selectivity of Peptoids as Azobenzene Scaffold for Molecular Solar Thermal Energy Storage.

Storing solar energy is a key challenge in modern science. MOlecular Solar Thermal (MOST) systems, in particular those based on azobenzene switches, have received great interest in the last decades. The energy storage properties of azobenzene (t1/2 <4 days; ΔH~270 kJ/kg) must be improved for future applications. Herein, we introduce peptoids as programmable supramolecular scaffolds to improve the energy storage properties of azobenzene-based MOST systems. We demonstrate with 3-unit peptoids bearing a single azobenzene chromophore that dynamics of the MOST systems can be tuned depending on the anchoring position of the photochromic unit on the macromolecular backbone. We measured a remarkable increase of the half-life of the metastable form up to 14 days at 20 °C for a specific anchoring site, significantly higher than the isolated azobenzene moiety, thus opening new perspectives for MOST development. We also highlight that liquid chromatography coupled to mass spectrometry does not only enable to monitor the different stereoisomers during the photoisomerization process as traditionally done, but also allows to determine the thermal back-isomerization kinetics.

Sidechain-Backbone Tetrel Bonding Interactions Provide a General Mechanism for trans-Peptoid Stabilization.

Cis-trans isomerization of amide bonds impedes de novo design of folded peptoids (poly-N-substituted glycines) with precise secondary structures and affects peptoid-biomolecule binding affinity. Herein, from X-ray, NMR and DFT studies of azapeptoids, we have discovered a tetrel bonding interaction that stabilizes trans-peptoids. We show that peptoids having α-heteroatoms and N-aryl groups in the sidechain adopt trans-amide geometries due to the presence of a nX /πAr →σ*Cα-N tetrel bonding interaction between the sidechain α-heteroatom lone pair (nX ) or π-electrons (πAr ) and the σ* orbital of the backbone Cα -N bond. Further, CD spectroscopic studies of oligo-proline host-guest model peptides showed that azapeptoid residues stabilize polyproline II helical conformation. These data indicate that the sidechain-backbone tetrel bonding could be leveraged to design peptoids with precise secondary structures for a wide range of biological and material applications.

A conjugate of chlorin e6 and cationic amphipathic peptoid: a dual antimicrobial and anticancer photodynamic therapy agent.

Cationic amphipathic structures are often utilized in natural membrane-active host-defense peptides. Negatively charged surface membranes of rapidly proliferating bacterial and cancer cells have been targeted by various synthetic peptides and peptidomimetics adopting the structural motif. Herein, we synthesized a set of conjugates composed of cationic amphipathic peptoids (i.e., oligo-N-substituted glycines) and a chlorin photosensitizer, named chlorin e6 (Ce6)-peptoid conjugates (CPCs). Among the nine CPCs, CPC 7, composed of Ce6, a PEG linker, and guanidine-rich helical amphipathic peptoids, exhibited a distinct photoresponsive inactivation of Gram-positive and Gram-negative bacteria. Subsequent studies showed that CPC 7 effectively killed various cancer cells after irradiation with red light (655 nm), suggesting the potential of CPC 7 as a dual antimicrobial and anticancer agent. Confocal laser scanning microscopy and flow cytometry data suggested that CPC 7 could induce apoptotic cell death. Our results show the potential of peptoid-based photosensitizer conjugates as a versatile platform for antimicrobial and anticancer photodynamic therapy agents and peptoid therapeutics.

Nα -Methylation of arginine: Implications for cell-penetrating peptides.

The field of cell-penetrating peptides is dominated by the use of oligomers of arginine residues. Octanol-water partitioning in the presence of an anionic lipid is a validated proxy for cell-penetrative efficacy. Here, we add one, two, or three N-methyl groups to Ac-Arg-NH2 and examine the effects on octanol-water partitioning. In the absence of an anionic lipid, none of these arginine derivatives can be detected in the octanol layer. In the presence of sodium dodecanoate, however, increasing N-methylation correlates with increasing partitioning into octanol, which is predictive of higher cell-penetrative ability. We then evaluated fully Nα -methylated oligoarginine peptides and observed an increase in their cellular penetration compared with canonical oligoarginine peptides in some contexts. These findings indicate that a simple modification, Nα -methylation, can enhance the performance of cell-penetrating peptides.

Recent advances in anticancer peptoids.

Since most tumors become resistant to drugs in a gradual and irreversible manner, making treatment less effective over time, anticancer drugs require continuous development. Peptoids are a class of peptidomimetics that can be easily synthesized and optimized. They exhibit a number of unique characteristics, including protease resistance, non-immunogenicity, do not interfere with peptide functionality and skeleton polarity, and can adopt different conformations. They have been studied for their efficacy in different cancer therapies, and can be considered as a promising alternative molecular category for the development of anticancer drugs. Herein, we discuss the extensive recent advances in peptoids and peptoid hybrids in the treatment of cancers such as prostate, breast, lung, and other ones, in the hope of providing a reference for the further development of peptoid anticancer drugs.

DNA origami protection and molecular interfacing through engineered sequence-defined peptoids.

DNA nanotechnology has established approaches for designing programmable and precisely controlled nanoscale architectures through specific Watson-Crick base-pairing, molecular plasticity, and intermolecular connectivity. In particular, superior control over DNA origami structures could be beneficial for biomedical applications, including biosensing, in vivo imaging, and drug and gene delivery. However, protecting DNA origami structures in complex biological fluids while preserving their structural characteristics remains a major challenge for enabling these applications. Here, we developed a class of structurally well-defined peptoids to protect DNA origamis in ionic and bioactive conditions and systematically explored the effects of peptoid architecture and sequence dependency on DNA origami stability. The applicability of this approach for drug delivery, bioimaging, and cell targeting was also demonstrated. A series of peptoids (PE1-9) with two types of architectures, termed as "brush" and "block," were built from positively charged monomers and neutral oligo-ethyleneoxy monomers, where certain designs were found to greatly enhance the stability of DNA origami. Through experimental and molecular dynamics studies, we demonstrated the role of sequence-dependent electrostatic interactions of peptoids with the DNA backbone. We showed that octahedral DNA origamis coated with peptoid (PE2) can be used as carriers for anticancer drug and protein, where the peptoid modulated the rate of drug release and prolonged protein stability against proteolytic hydrolysis. Finally, we synthesized two alkyne-modified peptoids (PE8 and PE9), conjugated with fluorophore and antibody, to make stable DNA origamis with imaging and cell-targeting capabilities. Our results demonstrate an approach toward functional and physiologically stable DNA origami for biomedical applications.

Unbiased peptoid cell screen identifies a peptoid targeting newly appeared cell surface vimentin on tumor transformed early lung cancer cells.

To identify potential new reagents and biomarkers for early lung cancer detection we combined the use of a novel preclinical isogenic model of human lung epithelial cells comparing non-malignant cells with those transformed to full malignancy using defined oncogenic changes and our on-bead two color (red and green stained cells) (OBTC) peptoid combinatorial screening methodology. The preclinical model used normal parent lung epithelial cells (HBEC3-KT, labeled with green dye) and isogenic fully malignant transformed derivatives (labeled with a red dye) via the sequential introduction of key genetic alterations of p53 knockdown, oncogenic KRAS and overexpression of cMYC (HBEC3p53, KRAS, cMYC). Using the unbiased OBTC screening approach, we tested 100,000 different peptoids and identified only one (named JM3A) that bound to the surface of the HBEC3p53, KRAS, cMYC cells (red cells) but not HBEC3-KT cells (green cells). Using the JM3A peptoid and proteomics, we identified the protein bound as vimentin using multiple validation approaches. These all confirmed the cell surface expression of vimentin (CSV) on transformed (HBEC3p53, KRAS, cMYC) but not on untransformed (HBEC3-KT) cells. JM3A coupled with fluorophores was able to detect and stain cell surface vimentin on very early stage lung cancers but not normal lung epithelial cells in a fashion comparable to that using anti-vimentin antibodies. We conclude: using a combined isogenic preclinical model of lung cancer and two color screening of a large peptoid library, we have identified differential expression of cell surface vimentin (CSV) after malignant transformation of lung epithelial cells, and developed a new peptoid reagent (JM3A) for detection of CSV which works well in staining of early stage NSCLCs. This new, highly specific, easy to prepare, CSV detecting JM3A peptoid provides an important new reagent for identifying cancer cells in early stage tumors as well as a resource for detection and isolating of CSV expressing circulating tumor cells.

Entry inhibition of hepatitis B virus using cyclosporin O derivatives with peptoid side chain incorporation.

Hepatitis B virus (HBV) infection is a serious worldwide health problem causing liver cirrhosis and hepatocellular carcinoma. The development of novel therapeutics targeting distinct steps of the HBV life cycle and combination therapy with approved drugs (i.e., nucleot(s)ides, interferon-α) are considered effective strategies for curing HBV. Among these strategies is the development of entry inhibitors that interfere with the host entry step of HBV to prevent viral infection and transmission. Herein, we generated a novel library of cyclosporin O (CsO) derivatives that incorporate peptoid side chains. Twenty-two CsO derivatives were evaluated for membrane permeability, cytotoxicity, and in vitro HBV entry inhibitory activity. The lead compound (i.e., compound 21) showed the greatest potency in the in vitro HBV entry inhibition assay (IC50 = 0.36 ± 0.01 µM) with minimal cytotoxicity. Our peptide-peptoid hybrid CsO scaffold can readily expand chemical diversity and is applicable for screening various targets requiring macrocyclic chemical entities.

Amphiphilic Peptoid-Directed Assembly of Oligoanilines into Highly Crystalline Conducting Nanotubes.

It is reported herein the synthesis of a novel amphiphilic diblock peptoid bearing a terminal conjugated oligoaniline and its self-assembly into small-diameter (D ≈ 35 nm) crystalline nanotubes with high aspect ratios (>30). It is shown that both tetraaniline (TANI)-peptoid and bianiline (BANI)-peptoid triblock molecules self-assemble in solution to form rugged highly crystalline nanotubes that are very stable to protonic acid doping and de-doping processes. The similarity of the crystalline tubular structure of the nanotube assemblies revealed by electron microscopy imaging, and X-ray diffraction analysis of the nanotube assemblies of TANI-functionalized peptoids and nonfunctionalized peptoids showed that the peptoid is an efficient ordered structure directing motif for conjugated oligomers. Films of doped TANI-peptoid nanotubes has a dc conductivity of ca. 95 mS cm-1 , while the thin films of doped un-assembled TANI-peptoids show a factor of 5.6 lower conductivity, demonstrating impact of the favorable crystalline ordering of the assemblies on electrical transport. These results demonstrate that peptoid-directed supramolecular assembly of tethered π-conjugated oligo(aniline) exemplify a novel general strategy for creating rugged ordered and complex nanostructures that have useful electronic and optoelectronic properties.

Advance in Hybrid Peptides Synthesis.

Hybrid peptides with heterogeneous backbone are a class of peptide mimics with adjustable proteolytic stability obtained from incorporating unnatural amino acid residues into peptide backbone. α/ß-peptides and peptide/peptoid hybrids are two types of hybrid peptides that are widely studied for diverse applications, and several synthetic methods have been developed. In this mini review, the advance in hybrid peptide synthesis is summarized, including solution-phase method, solid-phase method, and novel polymerization method. Conventional solution-phase method and solid-phase method generally result in oligomers with defined sequences, while polymerization methods have advantages in preparing peptide hybrid polymers with high molecular weight with simple operation and low cost. In addition, the future development of polymerization method to realize the control of the peptide hybrid polymer sequence is discussed.

Optimization of a cell surface vimentin binding peptoid to extract antagonist effect on lung cancer cells.

Targeting cytoskeletal proteins that are uniquely translocated to cancer cell surface may provide an alternative path for conventional drug discovery. Vimentin is such a cell surface-translocated cytoskeletal protein (CSV) found in non small cell lung cancer (NSCLC). We previously reported the identification of CSV-binding peptoid, named JM3A. While JM3A had no antagonist effect, here we used multiple strategies to optimize the binding of JM3A on CSV and extract the antagonistic effect. We first performed minimum pharmacophore identification studies using alanine/sarcosine scans. These studies revealed that residues 1-4 and 8 (from the C-terminus) are not important and those residues 5-7 are important for JM3A binding to CSV. We then found that our previous N-terminal benzophenone (BP)-coupled JM3A (JM3A-BP), which was used for pull-down and target identification studies, displayed 3-fold binding enhancement. The molecular docking studies indicated that the BP moiety binds to a new binding pocket on the vimentin coil 2 fragment, and further studies using 12 benzophenone-like moieties indicated that at least two phenyl groups are needed to occupy this new binding site. Interestingly, the binding was improved when non-important and bulky residues at the 4th and 8th positions were replaced with methyl groups (JM3A-4,8-BP). We next dimerized JM3A-4,8-BP to enhance the binding via the "avidity effect," using a central lysine linker to develop JM3A-4,8-BPD1 (EC50 = 300 nM). This showed 27- and 63-fold-improvement in binding over JM3A-4,8-BP and JM3A monomers, respectively. JM3A4,8BPD1 also displayed binding comparable to vimentin antibody. Finally, we observed an antagonist effect on H1299 NSCLC cell proliferation and viability from this most improved dimeric JM3A-4,8BPD1, which was not shown by the monomeric versions.

Atomic-level engineering and imaging of polypeptoid crystal lattices.

Rational design of supramolecular nanomaterials fundamentally depends upon an atomic-level understanding of their structure and how it responds to chemical modifications. Here we studied a series of crystalline diblock copolypeptoids by a combination of sequence-controlled synthesis, cryogenic transmission electron microscopy, and molecular dynamics simulation. This family of amphiphilic polypeptoids formed free-floating 2-dimensional monolayer nanosheets, in which individual polymer chains and their relative orientations could be directly observed. Furthermore, bromine atom side-chain substituents in nanosheets were directly visualized by cryogenic transmission electron microscopy, revealing atomic details in position space inaccessible by conventional scattering techniques. While the polypeptoid backbone conformation was conserved across the set of molecules, the nanosheets exhibited different lattice packing geometries dependent on the aromatic side chain para substitutions. Peptoids are inherently achiral, yet we showed that sequences containing an asymmetric aromatic substitution pattern pack with alternating rows adopting opposite backbone chiralities. These atomic-level insights into peptoid nanosheet crystal structure provide guidance for the future design of bioinspired nanomaterials with more precisely controlled structures and properties.

Nanoparticle-Mediated Assembly of Peptoid Nanosheets Functionalized with Solid-Binding Proteins: Designing Heterostructures for Hierarchy.

The fabrication of ordered architectures that intimately integrate polymer, protein, and inorganic components remains difficult. Two promising building blocks to tackle this challenge are peptoids, peptide mimics capable of self-assembly into well-defined structures, and solid-binding peptides, which offer a biological path to controlled inorganic assembly. Here, we report on the synthesis of 3.3-nm-thick thiol-reactive peptoid nanosheets from equimolar mixtures of unmodified and maleimide-derivatized versions of the Nbpe6Nce6 oligomer, optimize the location of engineered cysteine residues in silica-binding derivatives of superfolder green fluorescent protein for maleimide conjugation, and react the two components to form protein-peptoid hybrids exhibiting partial or uniform protein coverage on both of their sides. Using 10 nm silica nanoparticles, we trigger the stacking of these 2D structures into a multilayered material composed of alternating peptoid, protein, and organic layers. This simple and modular approach to hierarchical hybrid synthesis should prove useful in bioimaging and photocatalysis applications.

First series of N-alkylamino peptoid homooligomers: solution phase synthesis and conformational investigation.

The synthesis and conformational analysis of the first series of peptoid oligomers composed of consecutive N-(alkylamino)glycine units is investigated. We demonstrate that N-(methylamino)glycine homooligomers can be readily synthesized in solution using N-Boc-N-methylhydrazine as a peptoid submonomer and stepwise or segment coupling methodologies. Their structures were analyzed in solution by 1D and 2D NMR, in the solid state by X-ray crystallography (dimer 2), and implicit solvent QM geometry optimizations. N-(Methylamino)peptoids were found to preferentially adopt trans amide bonds with the side chain N-H bonds oriented approximately perpendicular to the amide plane. This orientation is conducive to local backbone stabilization through intra-residue hydrogen bonds but also to intermolecular associations. The high capacity of N-(methylamino)peptoids to establish intermolecular hydrogen bonds was notably deduced from pronounced concentration-dependent N-H chemical shift variation in 1H NMR and the antiparallel arrangement of mirror image molecules held together via two hydrogen bonds in the crystal lattice of dimer 2.